Primary cilia are ubiquitous antenna-like organelles that mediate cellular signaling and represent hotspots for human diseases termed ciliopathies. How signaling subcompartments are established within the microtubule-based organelle, and for example support Hedgehog or cGMP signal transduction pathways, remains a central question. Here we show that a C. elegans salt-sensing receptor type guanylate cyclase, GCY-22, accumulates at a high concentration within the distal region of the cilium. This receptor uses DAF-25 (Ankmy2 in mammals) to cross the transition zone (TZ) membrane diffusion barrier in the proximal-most region of the ciliary axoneme. Targeting of GCY-22 to the ciliary tip is dynamic, requiring the cargo-mobilizing intraflagellar transport (IFT) system. Disruption of transit across the TZ barrier or IFT trafficking causes GCY-22 protein mislocalization and defects in the formation, maintenance, and function of the ciliary tip compartment required for chemotaxis to low NaCl concentrations. Together, our findings reveal how a previously undescribed cilium tip cGMP signaling compartment is established and contributes to the physiological function of a primary cilium. signaling domains, remains limited, however.Cilia use two mechanisms, a trafficking system and a diffusion barrier, that function together to regulate the trafficking of proteins into, within, and out of cilia. The main ciliary trafficking machinery, intraflagellar transport (IFT), facilitates bidirectional transport of cargo, including signaling proteins, from the base/foundation (basal body) to the tip of the axoneme 2 . Anterograde IFT to the tip relies on kinesins, and cytoplasmic dynein enables retrograde transport back 3,4 . Two IFT modules, subcomplexes-A and -B 5,6 , together with another module containing BBS proteins (BBSome) that is thought to bridge the subcomplexes, play essential roles in cargo transport 7,8 . The best-known IFT cargos are axoneme structure components, including tubulin 9,10 , but signaling proteins, like the TRPV channel subunits OSM-9 and OCR-2 in the nematode C. elegans, are also transported 11 . Additionally, several mammalian ciliary signaling proteins, namely the GPCR SSTR3 and Hedgehog signaling component SMO, traverse the cilium by both IFT and diffusion 12 .To help confine proteins to cilia, a subdomain immediately distal to the basal body, called the transition zone (TZ), acts as a diffusion barrier for both membrane and soluble proteins [13][14][15] . How the TZ acts with IFT or other trafficking systems to regulate the composition of the sensory organelle is not well understood 16,17 .

13Primary cilia are ubiquitous antenna-like organelles that mediate cellular signaling and represent 14 hotspots for human diseases termed ciliopathies. How signaling subcompartments are established 15 within the microtubule-based organelle, and for example support Hedgehog or cGMP signal 16 transduction pathways, remains a central question. Here we show that a C. elegans salt-sensing 17 receptor type guanylate cyclase, GCY-22, accumulates at a high concentration within the distal region 18 of the cilium. This receptor uses DAF-25 (Ankmy2 in mammals) to cross the transition zone (TZ) 19 membrane diffusion barrier in the proximal-most region of the ciliary axoneme. Targeting of to the ciliary tip is dynamic, requiring the cargo-mobilizing intraflagellar transport (IFT) system. 21Disruption of transit across the TZ barrier or IFT trafficking causes GCY-22 protein mislocalization and 22 defects in the formation, maintenance, and function of the ciliary tip compartment required for 23 chemotaxis to low NaCl concentrations. Together, our findings reveal how a previously undescribed 24 cilium tip cGMP signaling compartment is established and contributes to the physiological function of 25 a primary cilium. 26 27 34 Cilia use two mechanisms, a trafficking system and a diffusion barrier, that function together to 35 regulate the trafficking of proteins into, within, and out of cilia. The main ciliary trafficking machinery, 36 intraflagellar transport (IFT), facilitates bidirectional transport of cargo, including signaling proteins, 37 from the base/foundation (basal body) to the tip of the axoneme 2 . Anterograde IFT to the tip relies on 38 kinesins, and cytoplasmic dynein enables retrograde transport back 3,4 . Two IFT modules, 39 subcomplexes-A and -B 5,6 , together with another module containing BBS proteins (BBSome) that is 40 thought to bridge the subcomplexes, play essential roles in cargo transport 7,8 . The best-known IFT 41 cargos are axoneme structure components, including tubulin 9,10 , but signaling proteins, like the TRPV 42 channel subunits OSM-9 and OCR-2 in the nematode C. elegans, are also transported 11 . Additionally, 43 several mammalian ciliary signaling proteins, namely the GPCR SSTR3 and Hedgehog signaling 44 component SMO, traverse the cilium by both IFT and diffusion 12 . 45 46To help confine proteins to cilia, a subdomain immediately distal to the basal body, called the 47 transition zone (TZ), acts as a diffusion barrier for both membrane and soluble proteins [13][14][15] . How the 48 TZ acts with IFT or other trafficking systems to regulate the composition of the sensory organelle is 49 not well understood 16,17 . 50 3 51 Signaling proteins can have different sub-ciliary localizations, including the proximal or distal 52 segments, or ciliary tip. For example, the C. elegans cyclic nucleotide gated channel TAX-2 localizes 53 to the proximal region adjoining the TZ 18 , while OSM-9 and OCR-2 11 and several GPCRs 18-20 localize 54 along the length of the cilium. In mammals, the kinesin-like pro...

Cell fate is maintained over long timescales, yet molecular fluctuations can lead to spontaneous loss of this differentiated state. Our simulations identified a possible mechanism that explains life-long maintenance of ASE neuron fate in C. elegans by the terminal selector transcription factor CHE-1. Here, fluctuations in CHE-1 level are buffered by the reservoir of CHE-1 bound at its target promoters, which ensures continued che-1 expression by preferentially binding the che-1 promoter. We provide experimental evidence for this mechanism by showing that che-1 expression was resilient to induced transient CHE-1 depletion, while both expression of CHE-1 targets and ASE function were lost. We identified a 130 bp che-1 promoter fragment responsible for this resilience, with deletion of a homeodomain binding site in this fragment causing stochastic loss of ASE identity long after its determination. Because network architectures that support this mechanism are highly conserved in cell differentiation, it may explain stable cell fate maintenance in many systems.

Cell fate is maintained over long timescales, yet molecular fluctuations can lead to spontaneous loss of this differentiated state. We uncovered a mechanism that explains lifelong maintenance of ASE neuron fate in C. elegans by the terminal selector transcription factor CHE-1. Fluctuations in CHE-1 level are buffered by the reservoir of CHE-1 bound at its target promoters, which ensure continued che-1 expression by preferentially binding the che-1 promoter. We validated this mechanism by showing that che-1 expression was resilient to induced transient CHE-1 depletion, while both expression of CHE-1 targets and ASE function were lost. We identified a 130 bp che-1 promoter fragment responsible for this resilience, with deletion of a homeodomain binding site in this fragment causing stochastic loss of ASE identity long after its determination. Because network architectures that support this mechanism are highly conserved in cell differentiation, it may explain stable cell fate maintenance in many systems. KeywordsNeuronal cell fate, bistability, gene regulatory network, stochastic gene expression, molecular fluctuations, homeodomain proteins, chemotaxis, C. elegans, terminal selector CHE-1, a transcription factor whose expression is transiently induced by the nuclear hormone receptor NHR-67 at the time of determination (Sarin et al. 2009). CHE-1 induces the expression of 500-1000 ASE-specific target genes, such as chemosensory receptors, ion-channels, and neuropeptides, by binding ASE motifs within their promoters (Etchberger et al. 2007). Its continued presence is required for expression of target genes after subtype determination (Etchberger et al. 2009). CHE-1 also upregulates its own expression. This positive feedback loop is necessary for sustaining che-1 expression and ASE cell fate directly after cell determination (Etchberger et al. 2007; Leyva-Diaz and Hobert 2019). Yet, it is unknown whether this positive feedback loop is sufficient for long-term maintenance of ASE fate. The impact of molecular noise, such as variability in CHE-1 protein copy number, on ASE fate maintenance has not been studied. Overall, it is an open question whether a reversible, bistable switch based on positive CHE-1 autoregulation would be sufficiently stable to maintain ASE fate for the animal's lifetime, or if additional mechanisms are necessary to ensure that, once ASE fate is determined, che-1 expression becomes independent of CHE-1 itself and can no longer spontaneously switch off.We show that sufficiently long, transiently induced depletion of CHE-1 caused permanent loss of ASE fate, indicating that it is controlled by a switch that remains reversible long after specification. This raises the question how the switch is protected against molecular noise, which could cause it to spontaneously lose ASE fate. Combining experimental measurements of the key parameters that control the magnitude of noise, i.e. the copy numbers and lifetimes of che-1 mRNA and protein, with stochastic models of the che-1 genetic network, revealed a novel...